Analysis of Genetic Variations in NSP4 of Type 1 Porcine Reproductive and Respiratory Syndrome Virus in China

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To investigate the prevalence and genetic variation of NSP4 in PRRSV-1 in China, 40 PRRSV whole-genomes sequences were collected and the NSP4 sequences were selected for nucleotide and amino acid homology analysis. In addition, amino acid site variation analysis was performed on the NSP4 sequences of the 40 PRRSV strains, and phylogenetic trees were constructed based on the whole-genomes and NSP4 sequences of 86 PRRSV strains. Results The nucleotide homology of NSP4 between 32 PRRSV-1 and 8 PRRSV-2 strains ranged from 57.3–62.4%, and the amino acid homology ranged from 58.6–64.5%. The nucleotide homology of NSP4 among the 32 PRRSV-1 strains ranged from 75.4–100.0%, and the amino acid homology ranged from 81.3–100.0%. Comparison of the NSP4 amino acid sequences of 32 PRRSV-1 and 8 PRRSV-2 strains revealed that the biggest difference was a deletion at the 83rd amino acid residue in PRRSV-1, and other differences manifested as varying degrees of mutations at multiple amino acid sites. Phylogenetic analysis showed that the PRRSV-1 strains isolated in China clustered into four subgroups of Subtype 1 Global: Amervac-like, BJEU06-1-like, HKEU16-like, and NMEU09-1-like strains. Conclusion PRRSV-1 NSP4 exhibits similarities to Subtype 1 Global strains and clusters into four subgroups. Although it differs from PRRSV-2 with a deletion in the 83rd amino acid residue, the catalytic triad amino acid residues are conserved. These findings have important implications for the development of secure and reliable detection methods and guide further research on PRRSV-1, providing a theoretical foundation for the development of new drugs and vaccines. PRRSV-1 PRRSV-2 NSP4 genetic variation phylogenetic analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious and severe infectious disease that can cause abortions, stillbirths, mummified fetuses, and respiratory diseases in pigs of all ages, particularly piglets [ 1 , 2 ]. The pathogen of PRRS is the porcine reproductive and respiratory syndrome virus (PRRSV), which is characterized by its ability to readily undergo variation and recombination, exhibit antibody-dependent enhancement, evade host immune responses, induce immune suppression, and provide insufficient cross-protection against heterologous strains. The introduction of new strains can lead to severe outbreaks [ 3 , 4 ]. This disease was initially reported in the United States in 1987, and it has since spread extensively to major pig-raising countries and regions worldwide, resulting in significant losses to the global pig industry [ 5 , 6 ]. In 1991, the Lelystad strain was first isolated and identified as a PRRSV in the Netherlands. In 1992, another strain, VR2332, was isolated in the United States. These two strains exhibited significant differences in their genomes sequences and were subsequently classified as porcine reproductive and respiratory syndrome virus-1 (PRRSV-1, European type) and porcine reproductive and respiratory syndrome virus-2 (PRRSV-2, American type), respectively [ 7 , 8 ]. In 2021, the International Committee on Taxonomy of Viruses updated the classification of the Arteriviridae family, and PRRSV was reclassified into the Betaarterivirus genus. PRRSV-1 is now considered a member of the Eurpobarte-virus subgenus and Betaarterivirussuid1 species, whereas PRRSV-2 is classified as a member of the Ampobartevirus subgenus and Betaarterivirussuid2 species. As a result of this reclassification, PRRSV-1 and PRRSV-2 are no longer considered two genotypes of the same species but are instead classified under different subgenera and species [ 9 ]. PRRSV-1 strains are widespread in many European countries, whereas PRRSV-2 strains are predominant in American and Asian countries. However, owing to the extensive pork trade within the European Union, the spread of PRRSV-1 among countries has accelerated, and the use of attenuated vaccines has increased the variation and evolution of PRRSV-1 strains [ 10 ]. Presently, PRRSV-1 has spread beyond Europe to countries such as the United States, Canada, South Korea, China, and Thailand [ 11 – 16 ]. Since the outbreak of PRRS in China, it has been dominated by sporadic epidemics of PRRSV-2, with few reports of PRRSV-1 cases. While most researchers believe that PRRSV-1 emerged in mainland China in 2011, the first PRRSV-1 strain (B13) was isolated in Beijing in 1997 and was identified as the earliest PRRSV-1 strain in China by mainland China customs. However, only its ORF5 gene was deposited in GenBank in 2004 (Accession No. AY633973) [ 17 ]. In 2011, Chen et al. reported two full genomes sequences of PRRSV-1, BJEU06-1 and NMEU09-1, which were the first wild-type PRRSV-1 strains isolated from mainland China [ 18 ]. The detection rate of PRRSV-1 in various regions of China is increasing, and PRRSV-1 has been detected in numerous provinces, including Fujian, Zhejiang, Guizhou, Hong Kong, Liaoning, Heilongjiang, and Henan. PRRSV-1 has spread to at least 22 provinces in China, including the central, northern, southern, eastern, northeastern, and southwestern regions (Fig. 1 ) [ 19 – 21 ]. Zhai et al. collected 750 samples from 50 breeding farms in Guangdong Province and found that the positivity rate of PRRSV-1 was 24.8% (186 samples), which is the highest reported positivity rate for PRRSV-1 in China [ 22 ]. Shi et al. performed a phylogenetic analysis of the ORF5 sequences deposited in GenBank and categorized PRRSV-1 into four subtypes: Subtype I Global, Subtype I Russian, Subtype II (Russia, Lithuania, and Belarus), and Subtype III [ 23 ]. Subtype I was previously prevalent in several European countries, particularly in Western Europe, and can be further divided into 12 evolutionary branches. It has now spread to continents beyond Europe. Subtypes II and III are predominantly found in Eastern Europe and Russia. Currently, most PRRSV-1 isolates in China belong to Subtype I Global. In 2017, Chen et al. first classified domestic PRRSV-1 into four genetic subgroups: Amervac-like, BJEU06-1-like, HKEU16-like, and NMEU09-1-like [ 24 ]. Among these, BJEU06-1 and NMEU09-1 were isolated from Beijing and Inner Mongolia, respectively. HKEU16 is a strain isolated in Hong Kong in 2007. Amervac is a vaccine strain produced by Laboratorios Hipra S.A. (HIPRA), a Spanish biopharmaceutical company [ 18 ]. As an immunosuppressive virus, PRRSV has developed numerous strategies to escape host antiviral innate immune responses [ 25 ]. Furthermore, PRRSV can target interferon-stimulated genes (ISGs) in order to subvert the host's antiviral responses, in addition to inhibiting interferon-beta (IFN-β) production [ 26 ]. PRRSV encodes nonstructural protein 4 (NSP4) that exhibits 3C-like serine proteinase (3CLSP) activity, featuring a conserved catalytic triad of His39-Asp64-Ser118. NSP4 is responsible for cleaving pp1a and pp1ab into multiple nonstructural proteins (NSP3–NSP12) [ 27 ]. As a crucial protease encoded by PRRSV, NSP4 plays a vital role in suppressing the host’s innate immune response. Apart from its ability to inhibit IFN-I signaling pathways, NSP4 also possesses the capability to activate the Notch signaling pathway, regulate antigen presentation pathways, and induce cell apoptosis [ 28 – 30 ]. These functions assist the virus in evading host immune responses effectively. NSP4 can interact with and cleave several host proteins, which may be the primary mechanism by which PRRSV evades host immune responses [ 30 , 31 ]. An in-depth investigation into the mechanism by which PRRSV NSP4 regulates host immune responses can facilitate a better understanding of PRRSV pathogenesis and the identification of its virulence factors, thereby providing new targets and a theoretical basis for weakening PRRSV and developing effective vaccines. 3CLSP, a primary protease involved in PRRSV replication, is a crucial target for drug design [ 32 ]. The majority of research on NSP4 protein has been conducted on PRRSV-2, resulting in limited investigation of PRRSV-1 NSP4. This study aimed to analyze 24 domestic strains of PRRSV-1 that were uploaded to GenBank. The NSP4 sequences of these strains were compared with typical strains from China and other countries, and their genetic variations were analyzed. This provides valuable data to support the study of PRRSV-1 NSP4 and valuable reference information for molecular epidemiological research on domestic PRRSV-1. Furthermore, this study provides a theoretical basis for effective prevention and control of PRRS. Methods Nucleotide homology analysis of PRRSV-1 NSP4 To gain insights into the origin and evolution of PRRSV-1 in China, 40 PRRSV whole-genomes sequences, comprising 32 PRRSV-1 sequences and 8 representative PRRSV-2 sequences, were retrieved from the GenBank database. Among these, 24 PRRSV-1 sequences from China were included, and the NSP4 gene was selected for sequence alignment analysis. The obtained NSP4 nucleotide sequences were analyzed for homology using the Clustal W method in the MegAlign function of the DNASTAR software package (version 7.0, Madison, WI). All the reference strain data were downloaded from the NCBI database (Table 1 ). Table 1 Information of the reference sequences for PRRSV Year Region Strain Accession no. Genotype 1991 Netherlands Lelystad virus M96262 PRRSV-1 1999 Netherlands MLV-DV KJ127878 PRRSV-1 2003 China HK3 KF287129 PRRSV-1 2004 China HK5 KF287130 PRRSV-1 2004 China HK8 KF287128 PRRSV-1 2004 China HK10 KF287131 PRRSV-1 2006 China BJEU06-1 GU047344 PRRSV-1 2007 China HKEU16 EU076704 PRRSV-1 2008 Belarus lena JF802085 PRRSV-1 2009 China SHE-2009 GQ461593 PRRSV-1 2009 China NMEU09-1 GU047345 PRRSV-1 2009 Spain Amervac PRRS GU067771 PRRSV-1 2010 Belarus SU1-Bel KP889243 PRRSV-1 2011 China GZ11-G1 KF001144 PRRSV-1 2011 China NVDC-FJ KC492506 PRRSV-1 2011 China NVDC-NM1-2011 JX187609 PRRSV-1 2011 China NVDC-NM2 KC492504 PRRSV-1 2011 China NVDC-NM3 KC492505 PRRSV-1 2012 China LNEU12 KM196101 PRRSV-1 2013 Russia WestSib13 KX668221 PRRSV-1 2014 Denmark Porcilis_DV-MLV MT311646 PRRSV-1 2014 China HLJB1 KT224385 PRRSV-1 2015 China FJEU13 KP860912 PRRSV-1 2015 China FJQEU14 KP860913 PRRSV-1 2015 China 15HEN1_EU KX967492 PRRSV-1 2016 China HENZMD-10 KY363382 PRRSV-1 2016 Russia Tyu16 MT008024 PRRSV-1 2018 China EUGDHD2018 MK639926 PRRSV-1 2018 China NPUST-2789-3W-2 MN242825 PRRSV-1 2020 China SC-2020-1 MW115431 PRRSV-1 2020 China TZJ226 OP566682 PRRSV-1 2020 China TZJ637 OP566683 PRRSV-1 1992 USA ATCC VR-2332 U87392 PRRSV-2 1996 China CH-1a AY032626 PRRSV-2 2000 China BJ-4 AF331831 PRRSV-2 2006 China TJ EU860248 PRRSV-2 2006 China JXA1 EF112445 PRRSV-2 2011 China QYYZ JQ308798 PRRSV-2 2013 China JL580 KR706343 PRRSV-2 2015 China HNyc15 KT945018 PRRSV-2 Homology analysis of PRRSV-1 NSP4 amino acids The 40 PRRSV-1 NSP4 nucleotide sequences were translated into their corresponding amino acid sequences, and their homology was analyzed using the Clustal W method in the MegAlign function of the DNASTAR software package. Analysis of the amino acid sequence of PRRSV-1 NSP4 By utilizing BioEdit software (version 7.2.6.1) for sequence alignment, differences in amino acid sites among the 40 PRRSV-1 NSP4 sequences were identified, allowing for a comprehensive assessment of the variation in the amino acid sequence of PRRSV-1 NSP4. Phylogenetic analysis of PRRSV-1 To gain a comprehensive understanding of the evolutionary relationship between the PRRSV-1 complete genomes and its NSP4 sequence, additional data were selected for phylogenetic analysis. A total of 86 PRRSV genomes sequences were obtained from GenBank, including 66 PRRSV-1 and 20 representative PRRSV-2 genomes sequences. The maximum likelihood method (ML) in the MEGA software (version 7.0, Center for Evolutionary Medicine and Informatics, Tempe, AZ) was used to construct a phylogenetic tree for genetic evolution analysis of the PRRSV-1 complete genomes or NSP4 sequence. Tree Of Life ( https://itol.embl.de ) was used to display, manipulate, and annotate the resulting phylogenetic tree [ 33 ]. Results Nucleotide homology analysis Nucleotide homology analysis was conducted on the NSP4 sequences of 40 PRRSV strains selected as reference strains. As shown in Fig. 2 , the nucleotide homology between the 32 PRRSV-1 NSP4 sequences and 8 PRRSV-2 NSP4 sequences ranged from 57.3–62.4%. The nucleotide homology between the 32 PRRSV-1 NSP4 sequences ranged from 75.4–100.0%. Notably, the highest homology (100.0%) was observed between the vaccine strains Porcilis DV-MLV and MLV-DV, suggesting that these strains originated from the same parental strain. Among the 24 PRRSV-1 NSP4 sequences isolated from China, high nucleotide homology was observed with NSP4 sequences of Subtype 1 Global strains (Lelystad virus, Amervac PRRS, Porcilis_DV-MLV, and MLV-DV), ranging from 84.4–98.5%. However, the nucleotide homology with the NSP4 sequences of other subtypes (Tyu16 of Subtype I Russian, WestSib13 of Subtype II, and Lena and SU1-Bel of Subtype III) was relatively low, ranging from 75.4–82.8%. Amino acid homology analysis The nucleotide sequences of NSP4 from the 40 PRRSV reference strains were translated into their corresponding amino acid sequences, and the resulting amino acid sequences were analyzed. As depicted in Fig. 3 , the amino acid homology between the 32 PRRSV-1 NSP4 sequences and 8 PRRSV-2 NSP4 sequences ranged from 58.6–64.5%. The amino acid homology among the 32 PRRSV-1 NSP4 sequences ranged from 81.3–100.0%. Among the 24 PRRSV-1 NSP4 sequences isolated from China, higher amino acid homology was observed with the NSP4 sequences of Subtype 1 Global strains (Lelystad virus, Amervac PRRS, Porcilis_DV-MLV, and MLV-DV), ranging from 88.2–100.0%. However, the amino acid homology with the NSP4 sequences of other subtypes (Tyu16 of Subtype I Russian, WestSib13 of Subtype II, and Lena and SU1-Bel of Subtype III) was relatively low, ranging from 81.3–88.7%. Amino acid sequence alignment BioEdit software was used to compare the amino acid sequences of the selected 40 representative PRRSV strains. As illustrated in Fig. 4 , the NSP4 sequence of PRRSV-1 comprises 203 amino acid residues, whereas that of PRRSV-2 comprises 204 amino acid residues. The most notable difference between the two was the deletion found at the 83rd amino acid residue of PRRSV-1 NSP4, whereas other differences were due to varying degrees of substitution at multiple amino acid residue sites. Notably, the histidine (His) at position 39, aspartic acid (Asp) at position 64, and serine (Ser) at position 118 are crucial amino acid residues for NSP4 to exert 3CLSP activity; these sites are relatively conserved without variation. Phylogenetic analysis To obtain a comprehensive understanding of the evolutionary relationship between Chinese PRRSV-1 and other PRRSV isolates, a total of 86 PRRSV genomes sequences were selected. This included 66 PRRSV-1 and 20 PRRSV-2, which were utilized for the construction of phylogenetic trees based on complete genomes and NSP4 sequences. Phylogenetic trees based on the complete genomes and NSP4 sequences are shown in Figs. 5 and 6 . All 24 reference sequences from China belonged to the Subtype 1 Global group, which was genetically closer to the vaccine strains and further from the other subtypes. Chinese PRRSV-1 isolates mainly clustered into four subgroups within the Subtype 1 Global group: Amervac-like, BJEU06-1-like, HKEU16-like, and NMEU09-1-like isolates. Discussion Currently, PRRSV-2 is the predominant strain in China. Although the detection rate of PRRSV-1 is lower than that of PRRSV-2, the mutation and evolution of PRRSV have accelerated in recent years, resulting in a sharp increase in the diversity of the virus. New and recombinant PRRSV strains are being detected more frequently. The detection rate of PRRSV-1 is increasing annually, and PRRSV-1 and PRRSV-2 commonly coexist in the same region [ 11 ]. PRRSV-related research in China has primarily focused on PRRSV-2, and the genetic evolution process of PRRSV-1 requires further investigation. To understand the genetic evolution of PRRSV-1 and its NSP4 sequence in China, this study aims to explore the prevalence of PRRSV-1 in China, compare 24 domestic PRRSV-1 NSP4 sequences with 8 representative PRRSV-1 and 8 PRRSV-2 NSP4 sequences, and analyze the NSP4 amino acid sequences of 40 representative strains to identify the relationship between the NSP4 amino acid sequences of PRRSV-1 and PRRSV-2. A total of 86 PRRSV strains were selected for a comprehensive analysis to construct phylogenetic trees based on whole-genomes and NSP4 sequences. The results showed that the nucleotide and amino acid homology of the NSP4 sequences of the Chinese PRRSV-1 and Subtype 1 Global representative strains were higher than those of other subtype representative strains. When comparing the NSP4 amino acid sequences of PRRSV-1 and PRRSV-2, the most significant difference was found at the 83rd amino acid residue of PRRSV-1, where a deletion was observed. Other differences were observed at multiple amino acid sites with varying degrees of substitutions. His at position 39, Asp at position 64, and Ser at position 118 are important amino acid sites for NSP4 to exert 3CLSP activity. These sites were relatively conserved with no observed variations, ensuring the normal function of the protein. NSP4 has 3CLSP activity, which plays a crucial role in viral replication. The catalytic triad of NSP4 comprises three amino acids, His-Asp-Ser, located at positions 39, 64, and 118. These three amino acids are essential for NSP4 to exert its 3CLSP activity. Mutations of these amino acids result in the loss of the protease activity of 3CLSP [ 34 ]. The Chinese PRRSV-1 NSP4 was analyzed for recombination using RDP4 and SimPlot software (version 3.5.1). According to the analysis results, there is no evidence of recombination events, which implies that Chinese PRRSV-1 strains have not undergone any recombination. The evolutionary trees constructed based on whole-genomes and NSP4 sequences revealed that Subtype 1 Global strains are prevalent in China. Chinese PRRSV-1 isolates were mainly clustered into four subgroups of Subtype 1 Global, which included Amervac-like, BJEU06-1-like, HKEU16-like, and NMEU09-1-like isolates. Compared with other subtypes, Subtype 1 Global PRRSV isolates are generally considered to have lower virulence and are primarily associated with reproductive disorders in sows. Clinical symptoms such as respiratory distress are only evident in case of co-infection with other pathogens. In China, the pathogenicity of PRRSV-1 has been studied extensively. The GZ11-G1 strain is considered moderately pathogenic, causing fever, high viral load, and lung and lymph node damage in piglets. High nucleotide sequence homology was found between GZ11-G1 and the Amervac vaccine strain through whole-genomes sequencing. This suggests that the use of attenuated live vaccines introduced through non-standard channels or the importation of vaccinated pigs may pose a risk of PRRSV infection in the pig population [ 19 ]. HLJB1 is another moderately pathogenic strain of PRRSV-1, causing short-term edema, conjunctival redness, cyanosis, and respiratory distress in infected pigs [ 35 ]. Recombination analysis revealed that the HLJB1 isolate resulted from a recombination event between the Amervac vaccine and the BJEU06-1 strain. This is the first report of natural recombination between PRRSV-1 vaccine strains and wild strains in China [ 24 ]. As international trade continues to expand, many pig farms in China may choose to irregularly introduce foreign breeds, thereby overcoming geographical restrictions. This raises concerns about the source of PRRSV-1 in China, which may be related to breed introduction. Therefore, it is essential to take appropriate measures to strengthen the monitoring and control of the spread and prevalence of PRRSV-1. Vaccination is an important tool for preventing and controlling PRRS in China; however, the effectiveness of PRRSV-1 vaccines requires further research. Additionally, attenuated vaccine strains are prone to regaining virulence, and the coexistence of low-pathogenicity vaccine strains and local wild strains on pig farms under immunological pressure leads to selective evolution between the vaccine strains and local wild strains. PRRSV-1 is not as widespread, infectious, diverse, or economically damaging as PRRSV-2. As a result, most studies have focused on PRRSV-2, neglecting the clinical detection and epidemiological investigation of PRRSV-1. These findings serve as a reminder to establish reliable and specific detection methods for European-type PRRS in China, and to formulate and continuously improve the corresponding diagnostic criteria. PRRSV is known to exhibit high mutation and recombination rates, making systematic phylogenetic and evolutionary analyses of specific genes, such as ORF5 and NSP2, and different nucleotide substitution rates between different sites, key areas of research [ 36 ]. Both NSP2 and ORF5 are highly variable, with ORF5 associated with neutralizing epitopes [ 37 ]. NSP2 and ORF5 are frequently used as target genes for molecular epidemiological monitoring of PRRSV [ 38 ]. The mechanism underlying PRRSV transmission can be explained from multiple perspectives. Analysis of genetic variations in NSP4 can provide a theoretical basis for understanding how NSP4 participates in regulating the host's innate response. Further research is needed to elucidate the role of NSP4 in the pathogenesis of PRRS, consolidate the genetic variation of PRRSV, and ultimately facilitate the prevention and control of PRRS [ 39 ]. By researching the genetic evolution of NSP4, it may be possible to pinpoint crucial amino acid sites, which could aid in the identification of significant antiviral targets and the development of innovative drug molecules. Additionally, it provides a theoretical basis for future vaccine development and the establishment of NSP4 detection methods. Conclusion The NSP4 nucleotide and amino acid sequences of Chinese PRRSV-1 were found to be highly similar to those of Subtype 1 Global representative strains, yet they displayed less similarity with those of other subtypes. PRRSV-1 NSP4 was found to have a deletion of the 83rd amino acid residue compared to PRRSV-2. The His-Asp-Ser catalytic triad site of NSP4 was relatively conserved, with no observed variations. Furthermore, no recombination events were detected in the NSP4 sequence of Chinese PRRSV-1. Phylogenetic trees based on the full genomes and NSP4 sequences indicated that Subtype 1 Global strains are prevalent in China, with Chinese PRRSV-1 isolates primarily clustered into four subgroups, namely Amervac-like, BJEU06-1-like, HKEU16-like, and NMEU09-1-like isolates. Declarations Acknowledgements We would like to thank Editage (www.editage.cn) for English language editing. Authors’ Contributions HS, HZ performed the experiments and wrote the manuscript; QL, YZ, GL analyzed the data; WK, LH, MZ conceived and designed the experiments; all authors read and approved the final manuscript. Funding This research was funded by the National Natural Science Foundation of China (31902279). Data Availability All datasets are available in the main manuscript. The dataset supporting the conclusions of this article is included within the article. Ethics approval and consent to participate All experiments were approved by the Institutional Animal Care and Use Committee of Foshan University. All authors read and approved the final manuscript. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. 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Infect Genet Evol. 2017;54:308-13. doi: 10.1016/j.meegid.2017.07.024. Huang C, Du Y, Yu Z, Zhang Q, Liu Y, Tang J, et al. Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Nsp4 Cleaves VISA to Impair Antiviral Responses Mediated by RIG-I-like Receptors. Sci Rep. 2016;6:28497. doi: 10.1038/srep28497. Wang TY, Sun MX, Zhang HL, Wang G, Zhan G, Tian ZJ, et al. Evasion of Antiviral Innate Immunity by Porcine Reproductive and Respiratory Syndrome Virus. Front Microbiol. 2021;12:693799. doi: 10.3389/fmicb.2021.693799. Ma Z, Wang Y, Zhao H, Xu AT, Wang Y, Tang J, et al. Correction: Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 4 Induces Apoptosis Dependent on Its 3C-Like Serine Protease Activity. PLoS One. 2020;15(2):e0230086. doi: 10.1371/journal.pone.0230086. Zhang F, Gao P, Ge XN, Zhou L, Guo X, Yang HC. Critical role of cytochrome c1 and its cleavage in porcine reproductive and respiratory syndrome virus nonstructural protein 4-induced cell apoptosis via interaction with nsp4. J Integr Agric. 2017;16(11):2573-85. doi: 10.1016/S2095-3119(17)61670-8. Chen J, Wang D, Sun Z, Gao L, Zhu X, Guo J, et al. Arterivirus nsp4 Antagonizes Interferon Beta Production by Proteolytically Cleaving NEMO at Multiple Sites. J Virol. 2019;93(12). doi: 10.1128/JVI.00385-19. Song Y, Guo Y, Li X, Sun R, Zhu M, Shi J, et al. RBM39 Alters Phosphorylation of c-Jun and Binds to Viral RNA to Promote PRRSV Proliferation. Front Immunol. 2021;12:664417. doi: 10.3389/fimmu.2021.664417. Guo R, Katz BB, Tomich JM, Gallagher T, Fang Y. Porcine Reproductive and Respiratory Syndrome Virus Utilizes Nanotubes for Intercellular Spread. J Virol. 2016;90(10):5163-75. doi: 10.1128/JVI.00036-16. Huang C, Zhang Q, Guo XK, Yu ZB, Xu AT, Tang J, et al. Porcine reproductive and respiratory syndrome virus nonstructural protein 4 antagonizes beta interferon expression by targeting the NF-kappaB essential modulator. J Virol. 2014;88(18):10934-45. doi: 10.1128/JVI.01396-14. Letunic I, Bork P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 2021;49(W1):W293-W6. doi: 10.1093/nar/gkab301. Ma Z, Wang Y, Zhao H, Xu AT, Wang Y, Tang J, et al. Porcine reproductive and respiratory syndrome virus nonstructural protein 4 induces apoptosis dependent on its 3C-like serine protease activity. PLoS One. 2013;8(7):e69387. doi: 10.1371/journal.pone.0069387. Ming S, Yongying M, Bohua L, Huiying L, Xiaoyu D, Qiaorong L, et al. Pathogenic Characterization of European Genotype Porcine Reproductive and Respiratory Syndrome Virus Recently Isolated in Mainland China. Open Virol J. 2017;11:83-9. doi: 10.2174/1874357901711010083. Frias-De-Diego A, Jara M, Pecoraro BM, Crisci E. Whole Genomes or Single Genes? A Phylodynamic and Bibliometric Analysis of PRRSV. Front Vet Sci. 2021;8:658512. doi: 10.3389/fvets.2021.658512. Li P, Shen Y, Wang T, Li J, Li Y, Zhao Y, et al. Epidemiological survey of PRRS and genetic variation analysis of the ORF5 gene in Shandong Province, 2020-2021. Front Vet Sci. 2022;9:987667. doi: 10.3389/fvets.2022.987667. Luo Q, Zheng Y, Zhang H, Yang Z, Sha H, Kong W, et al. Research Progress on Glycoprotein 5 of Porcine Reproductive and Respiratory Syndrome Virus. Animals (Basel). 2023;13(5). doi: 10.3390/ani13050813. Sha H, Zhang H, Luo Q, Zheng Y, Zhu Q, Wang N, et al. Variations in the NSP4 gene of the type 2 porcine reproductive and respiratory syndrome virus isolated in China from 1996 to 2021. Virus Genes. 2023;59(1):109-20. doi: 10.1007/s11262-022-01957-x. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 26 May, 2025 Read the published version in BMC Veterinary Research → Version 1 posted Editorial decision: Revision requested 25 Oct, 2024 Reviews received at journal 13 Jun, 2024 Reviews received at journal 12 Jun, 2024 Reviewers agreed at journal 11 Jun, 2024 Reviewers agreed at journal 10 Jun, 2024 Reviewers invited by journal 19 Jul, 2023 Editor invited by journal 19 Jul, 2023 Editor assigned by journal 18 Jul, 2023 Submission checks completed at journal 01 Jun, 2023 First submitted to journal 28 May, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2993449","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":206073550,"identity":"b2f6338b-af91-4332-82df-1c1953a46458","order_by":0,"name":"Huiyang Sha","email":"","orcid":"","institution":"School of Life Science and Engineering, Foshan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Huiyang","middleName":"","lastName":"Sha","suffix":""},{"id":206073551,"identity":"2e6e2d0a-1e59-4d5b-866b-9cd7d933b566","order_by":1,"name":"Hang Zhang","email":"","orcid":"","institution":"School of Life Science and Engineering, Foshan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hang","middleName":"","lastName":"Zhang","suffix":""},{"id":206073552,"identity":"f3af3339-8b10-4414-947f-1fa03bba7fcf","order_by":2,"name":"Qin Luo","email":"","orcid":"","institution":"School of Life Science and Engineering, Foshan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qin","middleName":"","lastName":"Luo","suffix":""},{"id":206073553,"identity":"cba50dd3-ab1b-40db-a7ba-1826222d3308","order_by":3,"name":"Yajie Zheng","email":"","orcid":"","institution":"School of Life Science and Engineering, Foshan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yajie","middleName":"","lastName":"Zheng","suffix":""},{"id":206073554,"identity":"5afd38ef-6750-4415-b77e-df82a0bcc700","order_by":4,"name":"Gan Li","email":"","orcid":"","institution":"School of Life Science and Engineering, Foshan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gan","middleName":"","lastName":"Li","suffix":""},{"id":206073555,"identity":"4b6082f6-729f-4af1-95ab-b42167478d33","order_by":5,"name":"Weili Kong","email":"","orcid":"","institution":"Gladstone Institutes of Virology and Immunology, University of California","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Weili","middleName":"","lastName":"Kong","suffix":""},{"id":206073556,"identity":"2366d879-2991-47a8-8bb1-cb6f7efa3662","order_by":6,"name":"Liangzong Huang","email":"","orcid":"","institution":"School of Life Science and Engineering, Foshan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Liangzong","middleName":"","lastName":"Huang","suffix":""},{"id":206073557,"identity":"003a5717-519e-4239-8079-adefa45792dc","order_by":7,"name":"Mengmeng Zhao","email":"data:image/png;base64,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","orcid":"","institution":"School of Life Science and Engineering, Foshan University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Mengmeng","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2023-05-29 03:29:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2993449/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2993449/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12917-025-04685-0","type":"published","date":"2025-05-26T15:57:35+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":38407817,"identity":"b8796b63-bacc-4218-b352-158d2407eb4c","added_by":"auto","created_at":"2023-06-12 14:58:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":59829,"visible":true,"origin":"","legend":"\u003cp\u003eGeographical distribution of PRRSV-1 in China. The white areas represent regions where PRRSV-1 has not been detected, while the blue areas denote regions with a low detection rate of PRRSV-1. The red areas indicate regions with a high detection rate of PRRSV-1.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-2993449/v1/28e2438b45a79cd49e6b0ce0.png"},{"id":38407820,"identity":"330550cd-89be-4822-8371-d1e43366a0ef","added_by":"auto","created_at":"2023-06-12 14:58:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":34349904,"visible":true,"origin":"","legend":"\u003cp\u003eNucleotide homology analysis of 40 PRRSV NSP4 sequences, consisting of 32 PRRSV-1 and 8 representative PRRSV-2 NSP4 sequences. Among these, the 32 PRRSV-1 NSP4 sequences were obtained from five typical foreign strains, three vaccine strains, and 24 domestically isolated strains with full genomes sequences uploaded for analysis.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-2993449/v1/38f15c9167d609c8f7792e17.png"},{"id":38407821,"identity":"5c7b8972-79df-4c2b-b351-f147fb1e7bdb","added_by":"auto","created_at":"2023-06-12 14:58:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":31518130,"visible":true,"origin":"","legend":"\u003cp\u003eHomology analysis of the amino acid sequence homology of 40 PRRSV NSP4 sequences, including 32 PRRSV-1 NSP4 sequences and 8 representative PRRSV-2 NSP4 sequences. Among these, the 32 PRRSV-1 NSP4 sequences were derived from five typical foreign strains, three vaccine strains, and 24 domestically isolated strains with complete genomes uploaded for analysis.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-2993449/v1/f1d0592ae2dbf94af04cccf5.png"},{"id":38407818,"identity":"2b289431-fd1f-407f-9df9-14d0ffbb6fbb","added_by":"auto","created_at":"2023-06-12 14:58:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1004186,"visible":true,"origin":"","legend":"\u003cp\u003eBioEdit software was utilized to conduct a comparative analysis of the amino acid sequences of different PRRSV NSP4. A total of 40 representative sequences available on GenBank were selected, with a focus on PRRSV-1 NSP4 sequences from China. The selected PRRSV-1 NSP4 sequences are highlighted in red, while the selected PRRSV-2 NSP4 sequences are shown in yellow. The conserved amino acids His39-Asp64-Ser118 of the 3CLSP active site are highlighted in green. A deletion at the 83\u003csup\u003erd\u003c/sup\u003e amino acid residue of PRRSV-1 NSP4 was identified when compared to PRRSV-2 NSP4, which is highlighted in red.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-2993449/v1/0f25bc89dd251bd33a0e0f7c.png"},{"id":38407822,"identity":"bf535f17-9513-4066-8cf1-7ee2ece6a30b","added_by":"auto","created_at":"2023-06-12 14:58:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":23524374,"visible":true,"origin":"","legend":"\u003cp\u003eMEGA 7 software was utilized to construct a phylogenetic tree based on complete genomes sequences of 66 PRRSV-1 isolates and 20 PRRSV-2 isolates. The maximum likelihood value was supported by the use of the general time reversible model (GTR), which has a gamma distribution with two constant positions (G+I) for the databases. Attenuated PRRSV-1 vaccine strains or vaccine derivatives are denoted by squares (■), while PRRSV-1 strains from China are represented by triangles (▲).\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-2993449/v1/4f08068e296869472ae2d394.png"},{"id":38407819,"identity":"d80e8b1c-3054-4bfd-8614-0a202910fc52","added_by":"auto","created_at":"2023-06-12 14:58:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":934801,"visible":true,"origin":"","legend":"\u003cp\u003eMEGA 7 software was utilized to construct a phylogenetic tree based on NSP4 sequences of 66 PRRSV-1 isolates and 20 PRRSV-2 isolates. The maximum likelihood value was supported by the use of the general time reversible model (GTR), which has a gamma distribution with two constant positions (G+I) for the databases. Attenuated PRRSV-1 vaccine strains or vaccine derivatives are denoted by squares (■), while PRRSV-1 strains from China are represented by triangles (▲).\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-2993449/v1/18d4f990b4a291f12b52bd95.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Analysis of Genetic Variations in NSP4 of Type 1 Porcine Reproductive and Respiratory Syndrome Virus in China","fulltext":[{"header":"Background","content":"\u003cp\u003ePorcine reproductive and respiratory syndrome (PRRS) is a highly contagious and severe infectious disease that can cause abortions, stillbirths, mummified fetuses, and respiratory diseases in pigs of all ages, particularly piglets [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The pathogen of PRRS is the porcine reproductive and respiratory syndrome virus (PRRSV), which is characterized by its ability to readily undergo variation and recombination, exhibit antibody-dependent enhancement, evade host immune responses, induce immune suppression, and provide insufficient cross-protection against heterologous strains. The introduction of new strains can lead to severe outbreaks [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This disease was initially reported in the United States in 1987, and it has since spread extensively to major pig-raising countries and regions worldwide, resulting in significant losses to the global pig industry [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In 1991, the Lelystad strain was first isolated and identified as a PRRSV in the Netherlands. In 1992, another strain, VR2332, was isolated in the United States. These two strains exhibited significant differences in their genomes sequences and were subsequently classified as porcine reproductive and respiratory syndrome virus-1 (PRRSV-1, European type) and porcine reproductive and respiratory syndrome virus-2 (PRRSV-2, American type), respectively [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In 2021, the International Committee on Taxonomy of Viruses updated the classification of the \u003cem\u003eArteriviridae\u003c/em\u003e family, and PRRSV was reclassified into the \u003cem\u003eBetaarterivirus\u003c/em\u003e genus. PRRSV-1 is now considered a member of the \u003cem\u003eEurpobarte-virus\u003c/em\u003e subgenus and \u003cem\u003eBetaarterivirussuid1\u003c/em\u003e species, whereas PRRSV-2 is classified as a member of the \u003cem\u003eAmpobartevirus\u003c/em\u003e subgenus and \u003cem\u003eBetaarterivirussuid2\u003c/em\u003e species. As a result of this reclassification, PRRSV-1 and PRRSV-2 are no longer considered two genotypes of the same species but are instead classified under different subgenera and species [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePRRSV-1 strains are widespread in many European countries, whereas PRRSV-2 strains are predominant in American and Asian countries. However, owing to the extensive pork trade within the European Union, the spread of PRRSV-1 among countries has accelerated, and the use of attenuated vaccines has increased the variation and evolution of PRRSV-1 strains [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Presently, PRRSV-1 has spread beyond Europe to countries such as the United States, Canada, South Korea, China, and Thailand [\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Since the outbreak of PRRS in China, it has been dominated by sporadic epidemics of PRRSV-2, with few reports of PRRSV-1 cases. While most researchers believe that PRRSV-1 emerged in mainland China in 2011, the first PRRSV-1 strain (B13) was isolated in Beijing in 1997 and was identified as the earliest PRRSV-1 strain in China by mainland China customs. However, only its \u003cem\u003eORF5\u003c/em\u003e gene was deposited in GenBank in 2004 (Accession No. AY633973) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In 2011, Chen et al. reported two full genomes sequences of PRRSV-1, BJEU06-1 and NMEU09-1, which were the first wild-type PRRSV-1 strains isolated from mainland China [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The detection rate of PRRSV-1 in various regions of China is increasing, and PRRSV-1 has been detected in numerous provinces, including Fujian, Zhejiang, Guizhou, Hong Kong, Liaoning, Heilongjiang, and Henan. PRRSV-1 has spread to at least 22 provinces in China, including the central, northern, southern, eastern, northeastern, and southwestern regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Zhai et al. collected 750 samples from 50 breeding farms in Guangdong Province and found that the positivity rate of PRRSV-1 was 24.8% (186 samples), which is the highest reported positivity rate for PRRSV-1 in China [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eShi et al. performed a phylogenetic analysis of the ORF5 sequences deposited in GenBank and categorized PRRSV-1 into four subtypes: Subtype I Global, Subtype I Russian, Subtype II (Russia, Lithuania, and Belarus), and Subtype III [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Subtype I was previously prevalent in several European countries, particularly in Western Europe, and can be further divided into 12 evolutionary branches. It has now spread to continents beyond Europe. Subtypes II and III are predominantly found in Eastern Europe and Russia. Currently, most PRRSV-1 isolates in China belong to Subtype I Global. In 2017, Chen et al. first classified domestic PRRSV-1 into four genetic subgroups: Amervac-like, BJEU06-1-like, HKEU16-like, and NMEU09-1-like [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Among these, BJEU06-1 and NMEU09-1 were isolated from Beijing and Inner Mongolia, respectively. HKEU16 is a strain isolated in Hong Kong in 2007. Amervac is a vaccine strain produced by Laboratorios Hipra S.A. (HIPRA), a Spanish biopharmaceutical company [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs an immunosuppressive virus, PRRSV has developed numerous strategies to escape host antiviral innate immune responses [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Furthermore, PRRSV can target interferon-stimulated genes (ISGs) in order to subvert the host's antiviral responses, in addition to inhibiting interferon-beta (IFN-β) production [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. PRRSV encodes nonstructural protein 4 (NSP4) that exhibits 3C-like serine proteinase (3CLSP) activity, featuring a conserved catalytic triad of His39-Asp64-Ser118. NSP4 is responsible for cleaving pp1a and pp1ab into multiple nonstructural proteins (NSP3\u0026ndash;NSP12) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. As a crucial protease encoded by PRRSV, NSP4 plays a vital role in suppressing the host\u0026rsquo;s innate immune response. Apart from its ability to inhibit IFN-I signaling pathways, NSP4 also possesses the capability to activate the Notch signaling pathway, regulate antigen presentation pathways, and induce cell apoptosis [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These functions assist the virus in evading host immune responses effectively. NSP4 can interact with and cleave several host proteins, which may be the primary mechanism by which PRRSV evades host immune responses [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. An in-depth investigation into the mechanism by which PRRSV NSP4 regulates host immune responses can facilitate a better understanding of PRRSV pathogenesis and the identification of its virulence factors, thereby providing new targets and a theoretical basis for weakening PRRSV and developing effective vaccines. 3CLSP, a primary protease involved in PRRSV replication, is a crucial target for drug design [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The majority of research on NSP4 protein has been conducted on PRRSV-2, resulting in limited investigation of PRRSV-1 NSP4. This study aimed to analyze 24 domestic strains of PRRSV-1 that were uploaded to GenBank. The NSP4 sequences of these strains were compared with typical strains from China and other countries, and their genetic variations were analyzed. This provides valuable data to support the study of PRRSV-1 NSP4 and valuable reference information for molecular epidemiological research on domestic PRRSV-1. Furthermore, this study provides a theoretical basis for effective prevention and control of PRRS.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eNucleotide homology analysis of PRRSV-1 NSP4\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo gain insights into the origin and evolution of PRRSV-1 in China, 40 PRRSV whole-genomes sequences, comprising 32 PRRSV-1 sequences and 8 representative PRRSV-2 sequences, were retrieved from the GenBank database. Among these, 24 PRRSV-1 sequences from China were included, and the \u003cem\u003eNSP4\u003c/em\u003e gene was selected for sequence alignment analysis. The obtained NSP4 nucleotide sequences were analyzed for homology using the Clustal W method in the MegAlign function of the DNASTAR software package (version 7.0, Madison, WI). All the reference strain data were downloaded from the NCBI database (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInformation of the reference sequences for PRRSV\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYear\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRegion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAccession no.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1991\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNetherlands\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLelystad virus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM96262\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNetherlands\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMLV-DV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKJ127878\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHK3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKF287129\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHK5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKF287130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHK8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKF287128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHK10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKF287131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBJEU06-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGU047344\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHKEU16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEU076704\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBelarus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003elena\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJF802085\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSHE-2009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGQ461593\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNMEU09-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGU047345\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmervac PRRS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGU067771\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBelarus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSU1-Bel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKP889243\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGZ11-G1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKF001144\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNVDC-FJ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKC492506\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNVDC-NM1-2011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJX187609\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNVDC-NM2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKC492504\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNVDC-NM3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKC492505\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLNEU12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKM196101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRussia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWestSib13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKX668221\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDenmark\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePorcilis_DV-MLV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMT311646\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHLJB1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKT224385\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFJEU13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKP860912\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFJQEU14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKP860913\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15HEN1_EU\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKX967492\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHENZMD-10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKY363382\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRussia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTyu16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMT008024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEUGDHD2018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMK639926\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNPUST-2789-3W-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMN242825\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSC-2020-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMW115431\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTZJ226\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOP566682\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTZJ637\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOP566683\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1992\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eATCC VR-2332\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eU87392\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1996\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCH-1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAY032626\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBJ-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAF331831\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTJ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEU860248\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJXA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEF112445\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eQYYZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJQ308798\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJL580\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKR706343\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHNyc15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKT945018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRRSV-2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eHomology analysis of PRRSV-1 NSP4 amino acids\u003c/h3\u003e\n\u003cp\u003eThe 40 PRRSV-1 NSP4 nucleotide sequences were translated into their corresponding amino acid sequences, and their homology was analyzed using the Clustal W method in the MegAlign function of the DNASTAR software package.\u003c/p\u003e\n\u003ch3\u003eAnalysis of the amino acid sequence of PRRSV-1 NSP4\u003c/h3\u003e\n\u003cp\u003eBy utilizing BioEdit software (version 7.2.6.1) for sequence alignment, differences in amino acid sites among the 40 PRRSV-1 NSP4 sequences were identified, allowing for a comprehensive assessment of the variation in the amino acid sequence of PRRSV-1 NSP4.\u003c/p\u003e\n\u003ch3\u003ePhylogenetic analysis of PRRSV-1\u003c/h3\u003e\n\u003cp\u003eTo gain a comprehensive understanding of the evolutionary relationship between the PRRSV-1 complete genomes and its NSP4 sequence, additional data were selected for phylogenetic analysis. A total of 86 PRRSV genomes sequences were obtained from GenBank, including 66 PRRSV-1 and 20 representative PRRSV-2 genomes sequences. The maximum likelihood method (ML) in the MEGA software (version 7.0, Center for Evolutionary Medicine and Informatics, Tempe, AZ) was used to construct a phylogenetic tree for genetic evolution analysis of the PRRSV-1 complete genomes or NSP4 sequence. Tree Of Life (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de\u003c/span\u003e\u003cspan address=\"https://itol.embl.de\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to display, manipulate, and annotate the resulting phylogenetic tree [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eNucleotide homology analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNucleotide homology analysis was conducted on the NSP4 sequences of 40 PRRSV strains selected as reference strains. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the nucleotide homology between the 32 PRRSV-1 NSP4 sequences and 8 PRRSV-2 NSP4 sequences ranged from 57.3\u0026ndash;62.4%. The nucleotide homology between the 32 PRRSV-1 NSP4 sequences ranged from 75.4\u0026ndash;100.0%. Notably, the highest homology (100.0%) was observed between the vaccine strains Porcilis DV-MLV and MLV-DV, suggesting that these strains originated from the same parental strain. Among the 24 PRRSV-1 NSP4 sequences isolated from China, high nucleotide homology was observed with NSP4 sequences of Subtype 1 Global strains (Lelystad virus, Amervac PRRS, Porcilis_DV-MLV, and MLV-DV), ranging from 84.4\u0026ndash;98.5%. However, the nucleotide homology with the NSP4 sequences of other subtypes (Tyu16 of Subtype I Russian, WestSib13 of Subtype II, and Lena and SU1-Bel of Subtype III) was relatively low, ranging from 75.4\u0026ndash;82.8%.\u003c/p\u003e\n\u003ch3\u003eAmino acid homology analysis\u003c/h3\u003e\n\u003cp\u003e \u003c/p\u003e \u003cp\u003eThe nucleotide sequences of NSP4 from the 40 PRRSV reference strains were translated into their corresponding amino acid sequences, and the resulting amino acid sequences were analyzed. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the amino acid homology between the 32 PRRSV-1 NSP4 sequences and 8 PRRSV-2 NSP4 sequences ranged from 58.6\u0026ndash;64.5%. The amino acid homology among the 32 PRRSV-1 NSP4 sequences ranged from 81.3\u0026ndash;100.0%. Among the 24 PRRSV-1 NSP4 sequences isolated from China, higher amino acid homology was observed with the NSP4 sequences of Subtype 1 Global strains (Lelystad virus, Amervac PRRS, Porcilis_DV-MLV, and MLV-DV), ranging from 88.2\u0026ndash;100.0%. However, the amino acid homology with the NSP4 sequences of other subtypes (Tyu16 of Subtype I Russian, WestSib13 of Subtype II, and Lena and SU1-Bel of Subtype III) was relatively low, ranging from 81.3\u0026ndash;88.7%.\u003c/p\u003e\n\u003ch3\u003eAmino acid sequence alignment\u003c/h3\u003e\n\u003cp\u003e \u003c/p\u003e \u003cp\u003eBioEdit software was used to compare the amino acid sequences of the selected 40 representative PRRSV strains. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the NSP4 sequence of PRRSV-1 comprises 203 amino acid residues, whereas that of PRRSV-2 comprises 204 amino acid residues. The most notable difference between the two was the deletion found at the 83rd amino acid residue of PRRSV-1 NSP4, whereas other differences were due to varying degrees of substitution at multiple amino acid residue sites. Notably, the histidine (His) at position 39, aspartic acid (Asp) at position 64, and serine (Ser) at position 118 are crucial amino acid residues for NSP4 to exert 3CLSP activity; these sites are relatively conserved without variation.\u003c/p\u003e\n\u003ch3\u003ePhylogenetic analysis\u003c/h3\u003e\n\u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo obtain a comprehensive understanding of the evolutionary relationship between Chinese PRRSV-1 and other PRRSV isolates, a total of 86 PRRSV genomes sequences were selected. This included 66 PRRSV-1 and 20 PRRSV-2, which were utilized for the construction of phylogenetic trees based on complete genomes and NSP4 sequences. Phylogenetic trees based on the complete genomes and NSP4 sequences are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. All 24 reference sequences from China belonged to the Subtype 1 Global group, which was genetically closer to the vaccine strains and further from the other subtypes. Chinese PRRSV-1 isolates mainly clustered into four subgroups within the Subtype 1 Global group: Amervac-like, BJEU06-1-like, HKEU16-like, and NMEU09-1-like isolates.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCurrently, PRRSV-2 is the predominant strain in China. Although the detection rate of PRRSV-1 is lower than that of PRRSV-2, the mutation and evolution of PRRSV have accelerated in recent years, resulting in a sharp increase in the diversity of the virus. New and recombinant PRRSV strains are being detected more frequently. The detection rate of PRRSV-1 is increasing annually, and PRRSV-1 and PRRSV-2 commonly coexist in the same region [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. PRRSV-related research in China has primarily focused on PRRSV-2, and the genetic evolution process of PRRSV-1 requires further investigation. To understand the genetic evolution of PRRSV-1 and its NSP4 sequence in China, this study aims to explore the prevalence of PRRSV-1 in China, compare 24 domestic PRRSV-1 NSP4 sequences with 8 representative PRRSV-1 and 8 PRRSV-2 NSP4 sequences, and analyze the NSP4 amino acid sequences of 40 representative strains to identify the relationship between the NSP4 amino acid sequences of PRRSV-1 and PRRSV-2. A total of 86 PRRSV strains were selected for a comprehensive analysis to construct phylogenetic trees based on whole-genomes and NSP4 sequences. The results showed that the nucleotide and amino acid homology of the NSP4 sequences of the Chinese PRRSV-1 and Subtype 1 Global representative strains were higher than those of other subtype representative strains. When comparing the NSP4 amino acid sequences of PRRSV-1 and PRRSV-2, the most significant difference was found at the 83rd amino acid residue of PRRSV-1, where a deletion was observed. Other differences were observed at multiple amino acid sites with varying degrees of substitutions. His at position 39, Asp at position 64, and Ser at position 118 are important amino acid sites for NSP4 to exert 3CLSP activity. These sites were relatively conserved with no observed variations, ensuring the normal function of the protein. NSP4 has 3CLSP activity, which plays a crucial role in viral replication. The catalytic triad of NSP4 comprises three amino acids, His-Asp-Ser, located at positions 39, 64, and 118. These three amino acids are essential for NSP4 to exert its 3CLSP activity. Mutations of these amino acids result in the loss of the protease activity of 3CLSP [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The Chinese PRRSV-1 NSP4 was analyzed for recombination using RDP4 and SimPlot software (version 3.5.1). According to the analysis results, there is no evidence of recombination events, which implies that Chinese PRRSV-1 strains have not undergone any recombination. The evolutionary trees constructed based on whole-genomes and NSP4 sequences revealed that Subtype 1 Global strains are prevalent in China. Chinese PRRSV-1 isolates were mainly clustered into four subgroups of Subtype 1 Global, which included Amervac-like, BJEU06-1-like, HKEU16-like, and NMEU09-1-like isolates.\u003c/p\u003e \u003cp\u003eCompared with other subtypes, Subtype 1 Global PRRSV isolates are generally considered to have lower virulence and are primarily associated with reproductive disorders in sows. Clinical symptoms such as respiratory distress are only evident in case of co-infection with other pathogens. In China, the pathogenicity of PRRSV-1 has been studied extensively. The GZ11-G1 strain is considered moderately pathogenic, causing fever, high viral load, and lung and lymph node damage in piglets. High nucleotide sequence homology was found between GZ11-G1 and the Amervac vaccine strain through whole-genomes sequencing. This suggests that the use of attenuated live vaccines introduced through non-standard channels or the importation of vaccinated pigs may pose a risk of PRRSV infection in the pig population [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. HLJB1 is another moderately pathogenic strain of PRRSV-1, causing short-term edema, conjunctival redness, cyanosis, and respiratory distress in infected pigs [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Recombination analysis revealed that the HLJB1 isolate resulted from a recombination event between the Amervac vaccine and the BJEU06-1 strain. This is the first report of natural recombination between PRRSV-1 vaccine strains and wild strains in China [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. As international trade continues to expand, many pig farms in China may choose to irregularly introduce foreign breeds, thereby overcoming geographical restrictions. This raises concerns about the source of PRRSV-1 in China, which may be related to breed introduction. Therefore, it is essential to take appropriate measures to strengthen the monitoring and control of the spread and prevalence of PRRSV-1. Vaccination is an important tool for preventing and controlling PRRS in China; however, the effectiveness of PRRSV-1 vaccines requires further research. Additionally, attenuated vaccine strains are prone to regaining virulence, and the coexistence of low-pathogenicity vaccine strains and local wild strains on pig farms under immunological pressure leads to selective evolution between the vaccine strains and local wild strains. PRRSV-1 is not as widespread, infectious, diverse, or economically damaging as PRRSV-2. As a result, most studies have focused on PRRSV-2, neglecting the clinical detection and epidemiological investigation of PRRSV-1. These findings serve as a reminder to establish reliable and specific detection methods for European-type PRRS in China, and to formulate and continuously improve the corresponding diagnostic criteria.\u003c/p\u003e \u003cp\u003ePRRSV is known to exhibit high mutation and recombination rates, making systematic phylogenetic and evolutionary analyses of specific genes, such as ORF5 and NSP2, and different nucleotide substitution rates between different sites, key areas of research [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Both NSP2 and ORF5 are highly variable, with ORF5 associated with neutralizing epitopes [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. NSP2 and ORF5 are frequently used as target genes for molecular epidemiological monitoring of PRRSV [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The mechanism underlying PRRSV transmission can be explained from multiple perspectives. Analysis of genetic variations in NSP4 can provide a theoretical basis for understanding how NSP4 participates in regulating the host's innate response. Further research is needed to elucidate the role of NSP4 in the pathogenesis of PRRS, consolidate the genetic variation of PRRSV, and ultimately facilitate the prevention and control of PRRS [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. By researching the genetic evolution of NSP4, it may be possible to pinpoint crucial amino acid sites, which could aid in the identification of significant antiviral targets and the development of innovative drug molecules. Additionally, it provides a theoretical basis for future vaccine development and the establishment of NSP4 detection methods.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe NSP4 nucleotide and amino acid sequences of Chinese PRRSV-1 were found to be highly similar to those of Subtype 1 Global representative strains, yet they displayed less similarity with those of other subtypes. PRRSV-1 NSP4 was found to have a deletion of the 83rd amino acid residue compared to PRRSV-2. The His-Asp-Ser catalytic triad site of NSP4 was relatively conserved, with no observed variations. Furthermore, no recombination events were detected in the NSP4 sequence of Chinese PRRSV-1. Phylogenetic trees based on the full genomes and NSP4 sequences indicated that Subtype 1 Global strains are prevalent in China, with Chinese PRRSV-1 isolates primarily clustered into four subgroups, namely Amervac-like, BJEU06-1-like, HKEU16-like, and NMEU09-1-like isolates.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Editage (www.editage.cn) for English language editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHS, HZ performed the experiments and wrote the manuscript; QL, YZ, GL analyzed the data; WK, LH, MZ conceived and designed the experiments; all authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the National Natural Science Foundation of China (31902279).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll datasets are available in the main manuscript. The dataset supporting the conclusions of this article is included within the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were approved by the Institutional Animal Care and Use Committee of Foshan University. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e School of Life Science and Engineering, Foshan University, Foshan, 528000, People\u0026apos;s Republic of China.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003e Gladstone Institutes of Virology and Immunology, University of California, San Francisco, 94158, USA.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhang H, Sha H, Qin L, Wang N, Kong W, Huang L, et al. 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Virus Genes. 2023;59(1):109-20. doi: 10.1007/s11262-022-01957-x.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"PRRSV-1, PRRSV-2, NSP4, genetic variation, phylogenetic analysis","lastPublishedDoi":"10.21203/rs.3.rs-2993449/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2993449/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePorcine reproductive and respiratory syndrome virus (PRRSV) is constantly evolving, and the detection rate of PRRSV-1 in China has been increasing annually. To investigate the prevalence and genetic variation of NSP4 in PRRSV-1 in China, 40 PRRSV whole-genomes sequences were collected and the NSP4 sequences were selected for nucleotide and amino acid homology analysis. In addition, amino acid site variation analysis was performed on the NSP4 sequences of the 40 PRRSV strains, and phylogenetic trees were constructed based on the whole-genomes and NSP4 sequences of 86 PRRSV strains.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe nucleotide homology of NSP4 between 32 PRRSV-1 and 8 PRRSV-2 strains ranged from 57.3\u0026ndash;62.4%, and the amino acid homology ranged from 58.6\u0026ndash;64.5%. The nucleotide homology of NSP4 among the 32 PRRSV-1 strains ranged from 75.4\u0026ndash;100.0%, and the amino acid homology ranged from 81.3\u0026ndash;100.0%. Comparison of the NSP4 amino acid sequences of 32 PRRSV-1 and 8 PRRSV-2 strains revealed that the biggest difference was a deletion at the 83rd amino acid residue in PRRSV-1, and other differences manifested as varying degrees of mutations at multiple amino acid sites. Phylogenetic analysis showed that the PRRSV-1 strains isolated in China clustered into four subgroups of Subtype 1 Global: Amervac-like, BJEU06-1-like, HKEU16-like, and NMEU09-1-like strains.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003ePRRSV-1 NSP4 exhibits similarities to Subtype 1 Global strains and clusters into four subgroups. Although it differs from PRRSV-2 with a deletion in the 83rd amino acid residue, the catalytic triad amino acid residues are conserved. These findings have important implications for the development of secure and reliable detection methods and guide further research on PRRSV-1, providing a theoretical foundation for the development of new drugs and vaccines.\u003c/p\u003e","manuscriptTitle":"Analysis of Genetic Variations in NSP4 of Type 1 Porcine Reproductive and Respiratory Syndrome Virus in China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-06-12 14:58:00","doi":"10.21203/rs.3.rs-2993449/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-25T07:13:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-13T06:43:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-13T03:48:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"128736667482880674169927076824862631584","date":"2024-06-11T06:55:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136813919348283444122636180245493790341","date":"2024-06-11T02:03:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-07-19T07:21:19+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2023-07-19T04:51:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-07-18T09:24:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-06-01T21:01:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2023-05-29T03:27:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"09b3c416-fa67-49e1-a790-7103650463fe","owner":[],"postedDate":"June 12th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-02T16:05:46+00:00","versionOfRecord":{"articleIdentity":"rs-2993449","link":"https://doi.org/10.1186/s12917-025-04685-0","journal":{"identity":"bmc-veterinary-research","isVorOnly":false,"title":"BMC Veterinary Research"},"publishedOn":"2025-05-26 15:57:35","publishedOnDateReadable":"May 26th, 2025"},"versionCreatedAt":"2023-06-12 14:58:00","video":"","vorDoi":"10.1186/s12917-025-04685-0","vorDoiUrl":"https://doi.org/10.1186/s12917-025-04685-0","workflowStages":[]},"version":"v1","identity":"rs-2993449","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2993449","identity":"rs-2993449","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}